Colorimetric sensors constructed of diacetylene materials

ABSTRACT

Colorimetric sensors comprising a receptor incorporated within polydiacetylene assemblies to form a transducer capable of indicating a color change when contacted with an analyte are disclosed. Methods of using the colorimetric sensor and a kit for the colorimetric detection of an analyte are also disclosed.

FIELD OF THE INVENTION

[0001] The present invention relates to a technique for detection ofanalytes using observable spectral changes in polydiacetyleneassemblies. More specifically, this invention relates to a colorimetricsensor comprising polydiacetylene assemblies and a method of using thesensor to detect an analyte.

BACKGROUND OF THE INVENTION

[0002] Diacetylenes are typically colorless and undergo additionpolymerization, either thermally or by actinic radiation. As thepolymerization proceeds, these compounds undergo a contrasting colorchange to blue or purple. When exposed to external stimuli such as heat,physical stress or a change of solvents or counterions, polydiacetylenesexhibit a further color changes produced by distortion of the planarbackbone conformation. Polydiacetylene assemblies are known to changecolor from blue to red with an increase in temperature or changes in pHdue to conformational changes in the conjugated backbone as described inMino, et al., Langmuir, Vol. 8, p. 594, 1992; Chance, et al., Journal ofChemistry and Physics, Vol. 71, 206, 1979; Shibutag, Thin Solid Films,Vol. 179, p. 433, 1989; Kaneko, et al., Thin Solid Films, Vol. 210, 548,1992; and U.S. Pat. No. 5,672,465. Utilization of this class ofcompounds is known for use as biochromic indicators as discussed in U.S.Pat. No. 5,622,872 and publication WO 02/00920.

[0003] Proposed applications of polydiacetylenes for detection ofanalytes are discussed in U.S. Pat. Nos. 6,395,561 B1; 6,306,598 B1;6,277,652; 6,183,722; 6,080,423 and publication WO 01/71317. Inparticular, attempts have been made to construct biosensors withreceptors that react specifically with pathogenic bacteria, viruses,toxins and the like incorporated into polydiacetylene membranes, and thecolor change (blue to red) is induced when the receptors bind to theirspecific analytes (pathogenic bacteria, viruses, toxins, etc.) Suchmethods require that the binding structure of the receptor and analytebe known, and the receptor identified. Synthesis of both the receptorsand the polydiacetylene membranes can be complicated and difficult. Thepolydiacetylene membranes can exhibit insufficient color change uponbinding with an analyte, requiring other substances for promoting thestructural change or enhancing analytical equipment to observe the colorchange.

SUMMARY OF THE INVENTION

[0004] A need continues to exist to make sensing devices employingdiacetylenes more accurate, more tailored to a given application, lesscomplex and more available to nontechnical personnel in a wide varietyof environments. Devices, which can be conveniently transported and usedindividually for a particular application then discarded, areparticularly desirable.

[0005] The present invention provides a calorimetric sensor to detectthe presence of analytes by spectral changes (color changes visible tothe naked eye or with a calorimeter) that occur as a result of thespecific binding of the analytes to polydiacetylene assemblies. Thepolydiacetylene assemblies indicate the presence of an analyte in asimple yet highly sensitive manner.

[0006] The present invention provides a colorimetric sensor comprising areceptor and the polymerization reaction product comprising at least onecompound of the formula

[0007] where R¹ is

[0008] R³, R⁸, R¹³, R²¹, R²⁴, R³¹ and R³³ are independently alkyl; R⁴,R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵, and R³² are independentlyalkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently alkylene, alkenylene,or arylene; R⁹ is alkylene or —NR³⁴—; R¹⁰, R¹², R²⁷, and R²⁹ areindependently alkylene or alkylene-arylene; R¹¹ and R²⁸ areindependently alkynyl; R¹⁷ is an ester-activating group; R²³ is arylene;R³⁰ is alkylene or —NR⁴—; R³⁴, and R³⁶ are independently H or C₁-C₄alkyl; p is 1-5; and n is 1-20; and where R¹ and R² are not the same.

[0009] Also provided is an easier preparation and use of polydiacetyleneassemblies in solution and for deposition on a substrate.

[0010] Also provided is a device and method for the detection of smallmolecules, pathogenic and non-pathogenic organisms, toxins, membranereceptors and fragments, volatile organic compounds, enzymes and enzymesubstrates, antibodies, antigens, proteins, peptides, nucleic acids, andpeptide nucleic acids.

[0011] Also provided is a simple to use, inexpensive test kit whosereliability is relativity stable in a wide range of environmentalconditions, and when the analyte is mixed with a number of othermaterials.

[0012] Also provided is a method for using the colorimetric sensor todetect an analyte by contacting the colorimetric sensor with an analyteand observing a color change.

[0013] Also provided is a method for indirect detection of an analyte bycontacting the calorimetric sensor with a probe that has an affinity forboth the analyte and the receptor, and observing no color change.

[0014] The above summary of the present invention is not intended todescribe each disclosed embodiment or every implementation of thepresent invention. The detailed description that follows moreparticularly exemplifies these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 shows a schematic representation of a colorimetric sensorof the present invention.

[0016]FIG. 2 shows a schematic representation of a calorimetric sensorarray of the present invention.

[0017]FIG. 3 shows a schematic representation of a colorimetric sensorfor contacting the sensor with an analyte.

[0018]FIG. 4 shows a schematic representation of a foldable colorimetricsensor for contacting the sensor with an analyte.

[0019]FIG. 5 shows a schematic representation of a colorimetric sensorof the present invention with one type of analyte delivery.

[0020]FIG. 6 shows a schematic representation of a calorimetric sensorarray of the prevent invention with one type of analyte delivery.

[0021]FIG. 7 is a phase diagram showing the colors of the coated anddried polydiacetylene film as a function of the contact angle of thesubstrate.

[0022]FIG. 8 is a phase diagram showing the colors of the coated anddried polydiacetylene film as a function of the surface tension of thesubstrate.

DETAILED DESCRIPTION

[0023] The present invention provides a colorimetic sensor comprisingdiacetylenic materials and a method of using the sensor to detect ananalyte. While the present invention is not so limited, an appreciationof various aspects of the invention will be gained through a discussionof the examples provided below.

[0024] For the following defined terms, these definitions shall beapplied, unless a different definition is given in the claims orelsewhere in this specification:

[0025] As used herein, the term “alkyl” refers to a straight or branchedchain or cyclic monovalent hydrocarbon radical having a specified numberof carbon atoms. Alkyl groups include those with one to twenty carbonatoms. Examples of “alkyl” as used herein include, but are not limitedto, methyl, ethyl, n-propyl, n-butyl, n-pentyl, isobutyl, and isopropyl,and the like. It is to be understood that where cyclic moieties areintended, at least three carbons in said alkyl must be present. Suchcyclic moieties include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyland cycloheptyl.

[0026] As used herein, the term “alkylene” refers to a straight orbranched chain or cyclic divalent hydrocarbon radical having a specifiednumber of carbon atoms. Alkylene groups include those with one tofourteen carbon atoms. Examples of “alkylene” as used herein include,but are not limited to, methylene, ethylene, trimethylene,tetramethylene and the like. It is to be understood that where cyclicmoieties are intended, at least three carbons in said alkylene must bepresent. Such cyclic moieties include cyclopropylene, cyclobutylene,cyclopentylene, cyclohexylene and cycloheptylene.

[0027] As used herein, the term “alkenylene” refers to a straight orbranched chain or cyclic divalent hydrocarbon radical having a specifiednumber of carbon atoms and one or more carbon—carbon double bonds.Alkenylene groups include those with two to eight carbon atoms. Examplesof “alkenylene” as used herein include, but are not limited to,ethene-1,2-diyl, propene-1,3-diyl, and the like.

[0028] As used herein, the term “arylene” refers to divalent unsaturatedaromatic carboxylic radicals having a single ring, such as phenylene, ormultiple condensed rings, such as naphthylene or anthrylene. Arylenegroups include those with six to thirteen carbon atoms. Examples of“arylene” as used herein include, but are not limited to,benzene-1,2-diyl, benzene-1,3-diyl, benzene-1,4-diyl,naphthalene-1,8-diyl, and the like.

[0029] As used herein, the term “alkylene-arylene”, refers to analkylene moiety as defined above bonded to an arylene moiety as definedabove. Examples of “alkylene-arylene” as used herein include, but arenot limited to, —CH₂-phenylene, —CH₂CH₂-phenylene, and—CH₂CH₂CH₂-phenylene.

[0030] As used herein, the term “alkynyl” refers to a straight orbranched chain or cyclic monovalent hydrocarbon radical having from twoto thirty carbons and at least one carbon-carbon triple bond. Examplesof “alkynyl” as used herein include, but are not limited to, ethynyl,propynyl and butynyl.

[0031] As used herein, the term “analyte(s)” refers to any material thatcan be detected by the sensor of the present invention. Such materialsinclude, but are not limited to small molecules, pathogenic andnon-pathogenic organisms, toxins, membrane receptors and fragments,volatile organic compounds, enzymes and enzyme substrates, antibodies,antigens, proteins, peptides, nucleic acids, and peptide nucleic acids.

[0032] As used herein, the term “bacteria” refers to all forms ofmicroorganisms considered to be bacteria including cocci, bacilli,spirochetes, sheroplasts, protoplasts, etc.

[0033] As used herein, the term “receptor” refers to any molecule with abinding affinity for an analyte of interest. Receptor includes, but isnot limited to, naturally occurring receptors such as surface membraneproteins, enzymes, lectins, antibodies, recombinant proteins, etc.;synthetic proteins; nucleic acids; c-glycosides; carbohydrates;gangliosides; and chelating agents.

[0034] As used herein, the terms “assembly”, or “self-assembly”, refersto any self-ordering of diacetylene molecules prior to polymerization.J. Israelachvili, Intermolecular and Surface Forces (2^(nd) Ed.),Academic Press, New York (1992), pp. 321-427.

[0035] As used herein, the term “self-assembling monolayer(s)” (SAMs)refers to any ordered ultrathin organic film formed on a given substrateby spontaneous self-ordering. A. Ulman, An Introduction to UltrathinOrganic Films, Academic Press, New York (1991), pp. 237-301.

[0036] As used herein, the term “transducer” describes a materialcapable of turning a recognition event at the molecular level into anobservable signal.

[0037] All numbers are herein assumed to be modified by the term“about.” The recitation of numerical ranges by endpoints includes allnumbers subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, and 5).

[0038] The present invention provides a calorimetric sensor comprisingnovel polydiacetylene assemblies incorporated with a receptor fordetection of an analyte. The polydiacetylene assemblies are polymerizedcompounds of the formula

[0039] where R¹ is

[0040] R³, R⁴, R¹³, R²¹, R²⁴, R³¹ and R³³ are independently alkyl; R⁴,R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵, and R³² are independentlyalkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently alkylene, alkenylene,or arylene; R⁹ is alkylene or —NR³⁴—; R¹⁰, R¹², R²⁷, and R²⁹ areindependently alkylene or alkylene-arylene; R¹¹ and R²⁸ areindependently alkynyl; R¹⁷ is an ester-activating group; R²³ is arylene;R³⁰ is alkylene or —NR³⁶—; R³⁴, and R³⁶ are independently H or C₁-C₄alkyl; p is 1-5; and n is 1-20; and where R¹ and R² are not the same.

[0041] Examples of R¹ when R¹ is alkyl include C₁-C₂₀ alkyl, C₆-C₁₈alkyl, and C₁₂-C₁₆ alkyl. Additional examples of R₁ when R¹ is alkylinclude dodecyl and hexadecyl.

[0042] Examples of R³ include C₁-C₂₀ alkyl, and C₆-C₁₈ alkyl. Additionalexamples of R³ include undecyl and pentadecyl.

[0043] Examples of R⁴ include C₁-C₁₄ alkylene, and C₁-C₄ alkylene.Additional examples of R⁴ include methylene (—CH₂—), trimethylene,(—CH₂CH₂CH₂—), and tetramethylene (—CH₂CH₂ CH₂CH₂—).

[0044] Examples of R⁵ include C₁-C₁₄ alkylene, and C₁-C₃ alkylene.Additional examples of R⁵ include ethylene (—CH₂CH₂—), and trimethylene(—CH₂CH₂CH₂—).

[0045] Examples of R⁶ when R⁶ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene. Additional examples of R⁶ when R⁶ is alkylene includeethylene (—CH₂CH₂—), and trimethylene (—CH₂CH₂CH₂—). Examples of R⁶ whenR⁶ is alkenylene include C₂-C₈ alkenylene, and C₂-C₄ alkenylene. Anadditional example of R when R⁶ is alkenylene includes ethenylene(—C═C—). Examples of R⁶ when R is arylene include C₆-C₁₃ arylene, andphenylene. An additional example of R⁶ when R⁶ is arylene isbenzene-1,2-diyl.

[0046] Examples of R⁷ include C₁-C₁₄ alkylene, and C₂-Cg alkylene.Additional examples of R⁷ include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂ CH₂CH₂—), pentamethylene(—CH₂CH₂CH₂CH₂CH₂—), hexamethylene (—CH₂CH₂CH₂ CH₂CH₂CH₂—),heptamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), octamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), and nonamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—).

[0047] Examples of R⁸ include Ct-C₁₋₆ alkyl, and C₁-C₈ alkyl. Additionalexamples of R⁸ include butyl, pentyl and hexyl.

[0048] R⁹ is independently alkylene or —NR³⁴—, where R³⁴ is H or C₁-C₄alkyl;

[0049] Examples of R⁹ when R⁹ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene, such as methylene (—CH₂—) for example. Examples of R⁹when R⁹ is —NR³⁴-include —NH—, —N(CH₂CH₃)—, and —N(CH₃)—.

[0050] Examples of R¹⁰ when R¹⁰ is alkylene include C₁-C₁₄ alkylene, andC₁-C₈ alkylene. Additional examples of R¹⁰ when R¹⁰ is alkylene includemethylene (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),tetramethylene (—CH₂CH₂ CH₂CH₂—), —C(CH₃)₂—, and —CH((CH₂)₁₄CH₃)—.Examples of R¹⁰ when R¹⁰ is alkylene-arylene include (C₁-C₁₄alkylene)-arylene, and (C₁-C₁₄ alkylene)-phenylene. An additionalexample of R¹⁰ when R¹⁰ is alkylene-arylene includes —CH₂-phenylene.

[0051] Examples of R¹¹ include C₂-C₃₀ alkynyl, and C₂₀-C₂₅ alkynyl.Additional examples of R¹¹ include C₂-C₃₀ alkynyl having at least twocarbon-carbon triple bonds (—C≡C—), and C₂₀-C₂₅ alkynyl having at leasttwo carbon-carbon triple bonds. Further examples of R¹¹ include C₂₂alkynyl having at least two carbon-carbon triple bonds, C₂₄ alkynylhaving at least two carbon-carbon triple bonds. Yet further examples ofR¹¹ include —(CH₂)₈—C═C—C≡C—(CH₂)₉CH₃, and —(CH₂)₈—C≡C—C≡C—(CH₂)₁₁CH₃.

[0052] Examples of R¹² when R¹² is alkylene include C₁-C₁₄ alkylene, andC₁-C₈ alkylene. Additional examples of R¹² when R¹² is alkylene includemethylene (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),tetramethylene (—CH₂CH₂ CH₂CH₂—), —C(CH₃)₂—, and —CH((CH₂)₁₄CH₃)—.Examples of R¹² when R¹² is alkylene-arylene include (C₁-C₁₄alkylene)-arylene, and (C₁-C₁₄ alkylene)-phenylene. An additionalexample of R¹² when R¹² is alkylene-arylene includes —CH₂-phenylene.

[0053] Examples of R¹³ include C₁-C₄ alkyl, such as methyl for example.

[0054] Examples of R¹⁴ include C₁-C₄ alkylene, such as ethylene(—CH₂CH₂—) for example.

[0055] Examples of R¹⁵ when R¹⁵ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene. Additional examples of R¹⁵ when R¹⁵ is alkylene includeethylene (—CH₂CH₂—), and trimethylene (—CH₂CH₂CH₂—). Examples of R¹⁵when R¹⁵ is alkenylene include C₂-C₈ alkenylene, and C₂-C₄ alkenylene.An additional example of R¹⁵ when R¹⁵ is alkenylene includes ethenylene(—C═C—). Examples of R¹⁵ when R¹⁵ is arylene include C₆-C₁₃ arylene, andphenylene. An additional example of R¹⁵ when R¹⁵ is arylene isbenzene-1,4-diyl.

[0056] Examples of R¹⁶ include C₁-C₁₄ alkylene, and C₂-C₉ alkylene.Additional examples of R¹⁶ include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂ CH₂CH₂—), pentamethylene(—CH₂CH₂CH₂CH₂CH₂—), hexamethylene (—CH₂CH₂CH₂ CH₂CH₂CH₂—),heptamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), octamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), and nonamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—).

[0057] Examples of R¹⁷ include groups that activate the neighboringester group toward acyl transfer. Such ester activating groups includepentafluorophenol, pentachlorophenol, 2,4,6-trichlorophenol,3-nitrophenol, N-hydroxysuccinimide, N-hydroxyphthalimide and thosedisclosed in M. Bodanszky, “Principles of Peptide Synthesis,”(Springer-Verlag 1984), for example. An additional example of R¹⁷ is2,5-dioxo-1-pyrrolidinyl.

[0058] Examples of R¹⁸ when R¹⁸ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene. Additional examples of R when R¹⁸ is alkylene includeethylene (—CH₂CH₂—), and trimethylene (—CH₂CH₂CH₂—). Examples of R¹⁸when R¹⁸ is alkenylene include C₂-C₈ alkenylene, and C₂-C₄ alkenylene.An additional example of R¹⁸ when R¹⁸ is alkenylene includes ethenylene(—C═C—). Examples of R¹⁸ when R¹⁸ is arylene include C₆-C₁₃ arylene, andphenylene. An additional example of R¹⁸ when R¹⁸ is arylene isbenzene-1,2-diyl.

[0059] Examples of R¹⁹ include C₁-C₁₄ alkylene, and C₂-C₉ alkylene.Additional examples of R¹⁹ include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂ CH₂CH₂—), pentamethylene(—CH₂CH₂CH₂CH₂CH₂—), hexamethylene (—CH₂CH₂CH₂ CH₂CH₂CH₂—),heptamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), octamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), and nonamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—).

[0060] Examples of R²⁰ include C₁-C₁₄ alkylene, C₁-C₉ alkylene, andC₁-C₄ alkylene. Additional examples of R²⁰ include methylene (—CH₂—),ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂CH₂CH₂—), pentamethylene (—CH₂CH₂CH₂CH₂CH₂—), hexamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), heptamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), octamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), and nonamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—).

[0061] Examples of R²¹ when R²¹ is alkyl include C₁-C₂₀ alkyl, C₆-C₁₈alkyl, and C₁₀-C₁₇ alkyl. Additional examples of R²¹ when R²¹ is alkylinclude decyl, undecyl, tridecyl, tetradecyl, pentadecyl, heptadecyl.

[0062] Examples of R²² include C₁-C₁₄ alkylene, and C₂-C₉ alkylene.Additional examples of R²² include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), and tetramethylene.

[0063] Examples of R²³ include C₆-C₁₃ arylene, and phenylene. Anadditional example of R²³ when R²³ is arylene is benzene-1,4-diyl.

[0064] Examples of R²⁴ include C₁-C₂₀ alkyl, and C₆-C₁₈ alkyl.Additional examples of R²⁴ include methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, and dodecyl.

[0065] Examples of R²⁵ include C₁-C₁₄ alkylene, and C₂-C₉ alkylene.Additional examples of R²⁵ include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂ CH₂CH₂—), pentamethylene(—CH₂CH₂CH₂CH₂CH₂—), hexamethylene (—CH₂CH₂CH₂ CH₂CH₂CH₂—),heptamethylene (—CH₂CH₂CH₂CH₂CH₂CH₂—), octamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—), and nonamethylene(—CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂CH₂—).

[0066] Examples of R²⁶ when R²⁶ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene. Additional examples of R²⁶ when R²⁶ is alkylene includeethylene (—CH₂CH₂—), and trimethylene (—CH₂CH₂CH₂—). Examples of R²⁶when R²⁶ is alkenylene include C₂-C₈ alkenylene, and C₂-C₄ alkenylene.An additional example of R²⁶ when R²⁶ is alkenylene includes ethenylene(—C═C—). Examples of R when R is arylene include C₆-C₁₃ arylene, andphenylene. An additional example of R²⁶ when R²⁶ is arylene isbenzene-1,2-diyl.

[0067] Examples of R²⁷ when R²⁷ is alkylene include C₁-C₁₄ alkylene, andC₁-C₈ alkylene. Additional examples of R²⁷ when R²⁷ is alkylene includemethylene (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),tetramethylene (—CH₂CH₂ CH₂CH₂—), —C(CH₃)₂—, and —CH((CH₂)₁₋₄CH₃)—.Examples of R²⁷ when R²⁷ is alkylene-arylene include (C₁-C₁₄alkylene)-arylene, and (C₁-C₁₄ alkylene)-phenylene. An additionalexample of R²⁷ when R²⁷ is alkylene-arylene includes —CH₂-phenylene.

[0068] Examples of R²⁸ include C₂-C₃₀ alkynyl, and C₂₀-C₂₅ alkynyl.Additional examples of R²⁸ include C₂-C₃₀ alkynyl having at least twocarbon-carbon triple bonds (—C≡C—), and C₂₀-C₂₅ alkynyl having at leasttwo carbon-carbon triple bonds. Further examples of R²⁸ include C₂₂alkynyl having at least two carbon-carbon triple bonds, C₂₄ alkynylhaving at least two carbon-carbon triple bonds. Yet further examples ofR²⁸ include —(CH₂)₈—C≡C—C≡C—(CH₂)₉CH₃, and —(CH₂)₈—C≡C—C≡C—(CH₂)₁₁CH₃.

[0069] Examples of R²⁹ when R²⁹ is alkylene include C₁-C₁₄ alkylene, andC₁-C₈ alkylene. Additional examples of R²⁹ when R²⁹ is alkylene includemethylene (—CH₂—), ethylene (—CH₂CH₂—), trimethylene (—CH₂CH₂CH₂—),tetramethylene (—CH₂CH₂ CH₂CH₂—), —C(CH₃)₂—, and —CH((CH₂)₁C₁₄CH₃)—.Examples of R²⁹ when R²⁹ is alkylene-arylene include (C₁-C₁₄alkylene)-arylene, and (C₁-C₁₄ alkylene)-phenylene. An additionalexample of R²⁹ when R²⁹ is alkylene-arylene includes —CH₂-phenylene.

[0070] R³⁰ is independently alkylene or —NR³⁶—, where R³⁶ is H or C₁-C₄alkyl;

[0071] Examples of R³⁰ when R³⁰ is alkylene include C₁-C₁₄ alkylene, andC₁-C₃ alkylene, such as methylene (—CH₂—) for example. Examples of R³⁰when R³⁰ is —NR³⁶-include —NH—, —N(CH₂CH₃)—, and —N(CH₃)—.

[0072] Examples of R³¹ include C₁-C₁₆ alkyl, and C₁-C₈ alkyl. Additionalexamples of R³¹ include butyl, pentyl and hexyl.

[0073] Examples of R³² include C₁-C₁₄ alkylene, and C₂-Cg alkylene.Additional examples of R³² include ethylene (—CH₂CH₂—), trimethylene(—CH₂CH₂CH₂—), tetramethylene (—CH₂CH₂ CH₂CH₂—), pentamethylene(—CH₂CH₂CH₂CH₂CH₂—), and hexamethylene (—CH₂CH₂CH₂ CH₂CH₂CH₂—).

[0074] Examples of R³³ include C₁-C₂₀ alkyl, C₆-C₁₈ alkyl, and C₁₀-C₁₆alkyl. Additional examples of R³³ include dodecyl, tetradecyl,hexadecyl, and octadecyl.

[0075] Compounds of the present invention also include those where p canbe 1 or 2 and n can be 1-20, 3-17, 6-14, or 9-11.

[0076] The invention is inclusive of the compounds described hereinincluding isomers, such as structural isomers and geometric isomers,salts, solvates, polymorphs and the like.

[0077] Preparation of the Diacetylene Compounds

[0078] Diacetylenes of the Formula XXIII can be prepared as outlined inScheme 1 where n is typically 1 to 4 and m is typically 10 to 14.

[0079] Compounds of formula XXIII can be prepared via oxidation fromcompounds of formula XXII by reaction with a suitable oxidizing agent ina suitable solvent such as DMF for example. Suitable oxidizing agentsinclude Jones reagent and pyridinium dichromate for example. Theaforesaid reaction is typically run for a period of time from 1 hour to48 hours, generally 8 hours, at a temperature from 0° C. to 40° C.,generally from 0° C. to 25° C.

[0080] Compounds of formula XXII can be prepared from compounds offormula XXI by reaction with a suitable acid chloride. Suitable acidchlorides include any acid chloride that affords the desired productsuch as lauroyl chloride, 1-dodecanoyl chloride, 1-tetradecanoylchloride, 1-hexadecanoyl chloride, and 1-octadecanoyl chloride forexample. Suitable solvents include ether, tetrahydrofuran,dichloromethane, and chloroform, for example. The aforesaid reaction istypically run for a period of time from 1 hour to 24 hours, generally 3hours, at a temperature from 0° C. to 40° C., generally from 0° C. to25° C., in the presence of a base such as trialkylamine or pyridinebase.

[0081] Compounds of formula XXI are either commercially available (e.g.where n is 1-4) or can be prepared from compounds of the formula XVIIIvia compounds XIX and XX as outlined in Scheme I and disclosed inAbrams, Suzanne R.; Shaw, Angela C. “Triple-bond isomerizations: 2- to9-decyn-1-ol,” Org. Synth. (1988), 66, 127-31 and Brandsma, L.“Preparative Acetylenic Chemistry,” (Elsevier Pub. Co.: New York, 1971),for example.

[0082] Diacetylenic compounds as disclosed herein can also be preparedby reacting compounds of formula XXII with an anhydride such assuccinic, glutaric, or phthalic anhydride in the presence of a suitablesolvents such as toluene. The aforesaid reaction is typically run for aperiod of time from 1 hour to 24 hours, generally 15 hours, at atemperature from 50° C. to 125° C., generally from 100° C. to 125° C.

[0083] The diacetylenic compounds as disclosed herein self assemble insolution to form ordered assemblies that can be polymerized using anyactinic radiation such as, for example, electromagnetic radiation in theUV or visible range of the electromagnetic spectrum. Polymerization ofthe diacetylenic compounds result in polymerization reaction productsthat have a color in the visible spectrum less than 570 nm, between 570nm and 600 nm, or greater than 600 nm depending on their conformationand exposure to external factors. Typically, polymerization of thediacetylenic compounds disclosed herein result in meta-stable blue phasepolymer networks that include a polydiacetylene backbone. Thesemeta-stable blue phase polymer networks undergo a color change frombluish to reddish-orange upon exposure to external factors such as heat,a change in solvent or counterion, if available, or physical stress forexample.

[0084] Polymerization products of some of the diacetylenic compoundsdisclosed herein can exhibit a reversible color change and/or a threestate color change. For example, after polymerization the resultingblue-phase polymer network can change color to a reddish-orange stateupon exposure to heat, a change in solvent or counterion, or physicalstress. This reddish-orange polymer network can then change color to ayellowish-orange state upon further exposure to heat, a change insolvent or counterion, or physical stress. Additionally, polymernetworks disclosed herein can cycle between these reddish-orange andyellowish-orange states in a reversible manner.

[0085] The ability of the diacetylenic compounds and theirpolymerization products disclosed herein to undergo a visible colorchange upon exposure to physical stress make them ideal candidates forthe preparation of sensing devices for detection of an analyte. Thepolydiacetylene assemblies formed from the disclosed diacetylenecompounds can function as a transducer in biosensing applications.

[0086] The structural requirements of a diacetylenic molecule for agiven sensing application are typically application specific. Featuressuch as overall chain length, solubility, polarity, crystallinity, andpresence of functional groups for further molecular modification allcooperatively determine a diacetylenic molecule's ability to serve as auseful sensing material. For example, in the case of blodetection of ananalyte in aqueous media, the structure of the diacetylenic compoundshould be capable of forming a stable dispersion in water, polymerizingefficiently to a colored material, incorporating appropriate receptorchemistry for binding to an analyte, and transducing that bindinginteraction by means of a color change. These abilities are dependent onthe structural features of the diacetylenic compounds.

[0087] The diacetylenic compounds of the present invention possess thecapabilities described above and can be easily and efficientlypolymerized into polydiacetylene assemblies that undergo the desiredcolor changes. Additionally, the diacetylenic compounds allow for theincorporation of large excesses of unpolymerizable material, such as areceptor described below, while still forming a stable, polymerizablesolution.

[0088] The disclosed diacetylenic compounds can be synthesized in arapid high-yielding fashion, including high-throughput methods ofsynthesis. The presence of functionality in the backbones of thediacetylenic compounds, such as heteroatoms for example, provides forthe possibility of easy structural elaboration in order to meet therequirements of a given sensing application. The diacetylenic compoundscan be polymerized into the desired polydiacetylene backbone containingnetwork by adding the diacetylene to a suitable solvent, such as waterfor example, sonicating the mixture, and then irradiating the solutionwith ultraviolet light, typically at a wavelength of 254 nm. Uponpolymerization the solution undergoes a color change to bluish-purple.Colorimetric Sensors comprising polydiacetylene assemblies Thecalorimetric sensors of the present invention comprising the discloseddiacetylene compounds can serve as the basis for the colorimetricdetection of a molecular recognition event in solution or coated on asubstrate. Such a molecular recognition device can be prepared by addinga receptor to the diacetylene monomer system either prior to or afterpolymerization. Upon polymerization or thereafter, the receptor iseffectively incorporated within the polymer network such thatinteraction of the receptor with an analyte results in a visible colorchange due to the perturbation of the conjugated ene-yne polymerbackbone.

[0089] In one embodiment, the receptor is physically mixed and dispersedamong the polydiacetylene assemblies. In an alternative embodiment, thereceptor is covalently bonded to the polydiacetylene assemblies.Examples of useful receptors include, but are not limited to surfacemembrane proteins, enzymes, lectins, antibodies, recombinant proteins,etc.; synthetic proteins; nucleic acids; c-glycosides; carbohydrates;gangliosides; and chelating agents. In one embodiment, the receptor is aphospholipid. In an alternate embodiment, the receptor is a glycerolincorporated into the diacetylene assembly by known methods such as thatdiscussed in Alcaraz, Marie-Lyne; Peng, Ling; Klotz, Phillipe; Goeldner,Maurice, J.Org.Chem. 1996, 61, 192-201.

[0090] The colometric sensors of the present invention formed from thedisclosed diacetylene compounds are amenable to a variety ofapplications that demand cost-effective, stable, accurate, consistentand quick diagnostics outside the laboratory setting. Applicationsinclude point-of-care testing, home testing diagnostics, military andindustrial detection of air —or water-borne pathogens and VOCs, and foodprocessing.

[0091] A sensor comprising the polydiacetylene assemblies can beobtained without the need to form a film by the conventional LB(Langmuir-Blodgett) process before transferring it onto an appropriatesupport. Alternatively, the polydiacetylene assemblies can be formed ona substrate using the known LB process as described in A. Ulman, AnIntroduction to Ultrathin Organic Films, Academic Press, New York(1991), pp. 101-219.

[0092] The present invention provides biosensing capabilities in adisposable adhesive product. The sensors are self-contained and do notrequire additional instrumentation to convey a measurable result.Alternatively, use with other analytical instrumentation is possible tofurther enhance sensitivity, such as fluorescence with the fluorescent“red” phase developed after detection of the analyte. The sensorsfunction to provide a rapid screening device when the detection of athreshold presence of a specific analyte is desired. Additionally, thesensors of the present invention are disposable and relativelyinexpensive.

[0093] In one embodiment of the invention, the calorimetric sensorcomprises a transducer formed from a receptor incorporated within thepolydiacetylene assemblies in solution. The solution can be provided ina simple vial system, with the analyte directly added to a vialcontaining a solution with the transducer specific to the analyte ofinterest. Alternatively, the colorimetric sensor could comprise multiplevials in a kit, with each vial containing a transducer comprisingpolydiacetylenes assemblies with incorporated receptors particular todifferent analytes. For those applications in which the analyte cannotbe added directly to the polydiacetylene transducer, a two-part vialsystem could be used. One compartment of the vial could contain reagentsfor sample preparation of the analyte physically separated from thesecond compartment containing the transducer formed from thepolydiacetylene assemblies. Once sample preparation is complete, thephysical barrier separating the compartments would be removed to allowthe analyte to mix with the transducer for detection.

[0094] In another embodiment of the present invention, the colorimetricsensor is a rapid indicator in a tape or label format as depicted inFIG. 1. FIG. 1 shows a tape or label 10 coated with a pressure sensitiveadhesive 20 and a transducer 30 coated on a substrate 40. Pressuresensitive adhesive 20 can affix tape or label 10 to a surface for directdetection of an analyte. Pressure sensitive adhesive 20 is isolated fromtransducer 30 containing the polydiacetylene assemblies to potentiallyminimize adverse effects. In FIG. 1, pressure sensitive adhesive 20surrounds the transducer 30 located in the center of tape or label 10.In an alternate embodiment (not shown), the pressure sensitive adhesiveand the transducer are combined.

[0095] Optionally, tape or label 10 will contain a transparent window onthe side of tape or label 10 that does not contain pressure sensitiveadhesive 20. The window would be centered under transducer 30 to allowthe user to view the color change without removing the tape or label 10from the surface containing the analyte.

[0096] In FIG. 2, the tape or label 110 is shown as array 111 composedof multiple transducers 112, 113, 114, 115, and 116. Each of transducers112, 113, 114, 115, and 116 could be formed from the same or differentpolydiacetylene assemblies with each polydiacetylene assemblyincorporating the same or different receptor. By varying transducers112, 113, 114, 115, and 116, array 111 can be designed to detectmultiple analytes at various concentration levels.

[0097] In another embodiment shown in FIG. 3, tape or label 100comprises a foldable substrate 101 with pressure sensitive adhesive 102on one side of foldable substrate 101, and transducer 103 placed on theopposing side of foldable substrate 101 facing pressure sensitiveadhesive 102. The surface containing a target analyte could be contactedwith pressure sensitive adhesive 102 to collect the sample. Once thesample containing the analyte is collected, the foldable substrate 101would be folded to contact the pressure sensitive adhesive 102 totransducer 103 as shown in FIG. 4. Optionally, foldable substrate 101could be perforated to allow separation of foldable substrate 101 intotwo or more parts, with one part containing pressure sensitive adhesive102 and another part containing the transducer 103. Both the foldablefeature and/or the perforations of foldable substrate 101 allow the userto prevent the transducer from contacting the sample surface thatcontains the analyte for applications requiring that functionality.

[0098] Optionally, foldable substrate 101 in FIG. 3 could also includemultiple transducers as shown in FIG. 2 and described above. Further,foldable substrate 101 could include a transparent window on the sideopposite transducer 103 for viewing any color change after foldablesusbstrate 101 is folded to contact transducer 103 to pressure sensitiveadhesive 102.

[0099] Alternative embodiments are shown in FIGS. 5 and 6 that allowdelivery of a fluid sample to the transducer via a microfluidic elementsuch as those described in U.S. Pat. Nos. 6,375,871 and 6,451,191. InFIG. 5, tape or label 301 contains a microfluidic element 302 thatdelivers the analyte to the transducer 303. In one application, pressuresensitive adhesive could be supplied on tape or label 301 on the sideopposite transducer 303 to allow the tape or label to be attached to asurface for storage or holding purposes such as attaching to a wall orcontainer.

[0100] In FIG. 6, a microfluidic element 401 is provided on tape orlabel 402. Microfluidic element 401 delivers the analyte to multiplewells 403, 404, 405, and 406 containing the same or differenttransducers. Each of the transducers in multiple wells 403, 404, 405,and 406 could be formed from the same or different polydiacetyleneassemblies with each polydiacetylene assembly incorporating the same ordifferent receptor. By varying the transducers, multiple wells 403, 404,405, and 406 can be designed to provide detection for multiple analytesat various concentration levels.

[0101] For those applications requiring sample preparation of theanalyte, a kit could contain a vial for reagant storage and mixing ofthe analyte before contacting the calorimetric sensor coated on atwo-dimensional substrate. In one embodiment, the kit could comprise avial for reagent storage and analyte preparation, with a cap systemcontaining the transducer of the present invention coated on asubstrate.

[0102] The present invention also provides a method for analysis of ananalyte, which comprises contacting the abovementioned colorimetricsensor with a solution sample or surface containing an analyte andutilizing an absorption measurement or a visual observation with thenaked eye to detect color change in the colorimetric sensor.

[0103] In an alternative embodiment, the present invention provides amethod for indirect detection of an analyte by selection of a probe withan affinity to bind with both the receptor incorporated into thepolydiacetylene assemblies and the analyte. The probe selected willdemonstrate a competitive affinity with the analyte. When the analyte ofinterest is present, the probe will bind to the analyte rather than thereceptor on the polydiacetylene backbone, resulting in no color change.If the analyte is absent, the probe will bind to the receptorincorporated on the polydiacetylene backbone, resulting in a colorchange from blue to red. The probe can contact the transducer after theanalyte contacts the transducer, or can be mixed with the analyte priorto the mixture contacting the transducer.

[0104] The probe can be contacted with the transducer in solution orcoated on a substrate. The probe will be any molecule with an affinityfor both the target analyte and the receptor. Possible probes for use inthe present invention include membrane disrupting peptides such asalamethicin, magainin, gramicidin, polymyxin B sulfate, and melittin.

[0105] Using the indirect method of detection, high detection levels arepossible based on the concentration of probe used. For detectionstrategy, probe concentrations can be chosen to correspond to desiredconcentration levels of detection. The method of indirect detectionusing the probe allows design of the system around the type andconcentration of the probe for desired sensitivity in a givenapplication. This allows the transducer to be universal to multipleanalytes of interest. For example, a single transducer(polydiacetylene/receptor combination) could serve to detect multipleanalytes by varying the probe in contact with the transducer inaccordance with the probe's affinity for the analyte.

[0106] Substrate Characteristics

[0107] The substrates of the present invention can be characterized bycontact angle measurements using milli-Q (Millipore) water and methyleneiodide (Aldrich) probe liquids. For a contact angle measurement, agoniometer from Rame-Hart is used to measure the contact angle that adrop of a probe liquid forms when placed on a substrate. Although thedrop covers a macroscopic area of the substrate, the interaction of theliquid and the surface probes only the outermost 1-5 Ångstroms of thesurface. Thus, contact angle analysis provides an accurate and sensitivetechnique for characterizing surface energetics as discussed in A.Ulman, An Introduction to Ultrathin Organic Films, Academic Press, NewYork (1991), pp. 48-58.

[0108] The coating substrates used in the present invention can beconsidered to encompass two broad categories. The first of thesecategories include highly flat substrates, such as evaporated gold onatomically flat silicon (111) wafers, atomically flat silicon (111)wafers, or float glass, which are bare and modified with self-assemblingmonolayers (SAMs) to alter their surface energy in a systematic fashion.The second class of surfaces comprised surfaces with a highly texturedtopography that included many different classes of materials rangingfrom paper substrates to polymeric ink receptive coatings to structuredpolymeric films, microporous films, and membrane materials. Commoncharacteristics amongst these substrates are the large surface roughnessand/or porosity. In these highly textured surfaces, the measurements ofcontact angles and the determination of polar and dispersive surfaceenergies from these contact angle measurements cannot be regarded as anequilibrium characterization of their true thermodynamic energies. Forpurposes of the present invention, the contact angles indicate an“effective” or “practical” surface energy that can be used to classifythese substrates for comparative purposes.

[0109] TABLE 1 summarizes the substrates coated with the polydiacetyleneassemblies for use as a calorimetric sensor of the present invention.The SAMs used to modify the substrate are listed with the substrate whenused. The substrate contact angles as measured with water and methyleneiodide are shown, as well as the dispersive and polar components of thesurface energy calculated by the Geometric Mean Method as shown in S.Wu; Polymer Interface and Adhesion; Marcel Dekker, New York (1982). Thelast column lists the color of the dry PDA coating. TABLE 1 Adv. θ Adv.θ γ γ (Polar) Color of (Water) (MI) (Dispersive) (dynes/ Dry NumberSubstrate Manufacturer (°) (°) (dynes/cm) cm) Coating 1 Reverse Aldrich;158 ± 3.0 <5 101.7 39.5 Blue Chromatography Milwaukee, WI Si Gel Plate 2Manila Folder Smead, No. 2- 115 ± 5.0 <5 76.2 7.9 Blue Paper 153L-2,Hastings, MN 3 Clean Float Corning Glass  90 ± 3.8 42 ± 2.4 39.6 0.9Blue Glass Works; Corning, NY 4 Textured Photo 3M Part No. 34- 123 ± 5.4<5 82.1 13.3 Blue Paper 8506-6373-2; St. Paul, MN 5 Gloss Photo 3M PartNo. 34- 105 ± 8.0 30 ± 1.3 58.1 1.5 Blue Paper 8506-6378-1; St. Paul, MN6 PE IR Card 3M Type 61- 144 ± 0.6 <5 95.4 30.0 Blue 100-12; St. Paul,MN 7 PTFE IR Card 3M Type 62; St. 133 ± 2.3 66 ± 2.5 44.0 7.6 Blue Paul,MN 8 Octadecyltri- Gelest, Inc.; 112 ± 1.1 69 ± 1.9 28.3 0.1 Redchlorosilane Morrisville, PA SAM on Si (111) 9 Perfluorodecyl- Gelest,Inc.; 113 ± 1.6 91 ± 1.3 11.7 0.7 Red 1H, 1H, 2H, 2H- Morrisville, PAtrichlorosilane on Si (111) Wafer 10 Octadecyltri- Gelest, Inc.; 112 ±2.2 65 ± 1.8 31.9 0.3 Red chlorosilane Morrisville, PA SAM on Si (111)11 Dodecanethiol Aldrich; 108 ± 3.0 65 ± 4.4 30.0 0 Red SAM onMilwaukee, WI Evaporated Au 12 Octadecanethiol Aldrich; 107 ± 0.6 67 ±2.7 27.7 0 Red SAM on Milwaukee, WI Evaporated Au 13 Microstructured 3Msample No. 155 ± 4.5 110 ± 6.0  9.8 2.5 Red PP PP-3445; St. Paul, MN 14FC TIPS 3M sample; St. 112 ± 5.5 127 ± 11.5 0.1 8.7 Red Membrane Paul,MN 15 PVDF Membrane Millipore  86 ± 2.9 61 ± 1.1 22.8 5.4 RedCorporation XF1J076T8; Bedford, MA 16 Bare Si (111) Siltec  52 ± 2.6 47± 2.8 18.7 29.2 Mixed Wafer Corporation; Salem, OR 17 Bare Evaporated 3MSample, St.  97 ± 4.5 48 ± 0.6 38.9 0.1 Mixed Au Paul, MN 1811-Mercapto- Aldrich;  58 ± 1.6 38 ± 1.7 26.0 19.8 Mixed 1-undecanol onMilwaukee, WI Evaporated Au 19 16-Mercapto- Aldrich;  40 ± 1.6 35 ± 6.021.4 35.7 Mixed hexadecanoic on Milwaukee, WI Evaporated Au

[0110]FIGS. 6 and 7 show the phase diagram based on the colorimetricobservations of the dried coatings and the contact angle analysis of thecoated substrates. FIG. 6 shows the resulting color of the coatedsubstrate as a function of advancing contact angle of water versusadvancing contact angle of methylene iodide. FIG. 7 shows the resultingcolor of the coated substrate as a function of the polar component ofthe substrate surface energy versus the dispersive component of thatsurface energy as calculated by the Geometric Mean method from theadvancing contact angles of water and methylene iodide as provided in S.Wu; Polymer Interface and Adhesion; Marcel Dekker, New York (1982). Thesurfaces for which the polydiacetylene coating remained in its initialblue color are identified by a filled-in circle on each plot. Thesurfaces identified with a circle are those on which the initial “blue”phase transformed into the red phase upon drying. Finally, thetriangular points identify surfaces on which the dry coating showed amixture of the blue and red phases.

[0111] The numbers in TABLE 1 assigned to the various substratescorrelate to the symbols in FIGS. 6 and 7.

[0112] In one embodiment of the invention that maintains the original“blue” phase of the polydiacetylene assemblies upon drying, the coatedsubstrates exhibit advancing contact angles with methylene iodide below50° as depicted in FIG. 6. This condition corresponds to substratescharacterized by a dispersive component of their surface energy greaterthan 40 dynes/cm on FIG. 7. For applications requiring the retention ofthe original “blue” phase, topography and surface energy will impact theeffectiveness of the transducer on the substrate in characteristics suchas color contrast at detection and shelf-life.

[0113] In an alternate embodiment, substrates with these properties thathave an advancing contact angle with water less than 90° result in drycoatings containing a mixture of the blue and red phases as depicted inFIG. 6. This condition would correspond to surfaces in which thedispersive surface energy component could be less than 40 dynes/cm butwith a polar surface energy component greater than at least 10 dynes/cmin FIG. 7.

EXAMPLES

[0114] The present invention should not be considered limited to theparticular examples described below, but rather should be understood tocover all aspects of the invention as fairly set out in the attachedclaims. Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification. Allparts, percentages, ratios, etc. in the examples and the rest of thespecification are by mole unless indicated otherwise. All solvents andreagents without a named supplier were purchased from Aldrich Chemical;Milwaukee, Wis. Water was purified by the use of a U-V Milli-Q waterpurifier with a resistivity of 18.2 Mohms/cm. (Millipore, Bedford Mass.)

[0115] For those diacetylene mixtures that were probe sonicated, a rangeof samples were prepared by separating the initial diacetylene solutioninto several vials for probe sonication over a range of power and timesto determine the settings that induce successful self-assembly in thediacetylene monomers. Although probe sonication was done at a powersetting of 5 for 1 minute in the following examples, one skilled in theart would know that appropriate adjustments in power and time canempirically create the same result.

[0116] Colorimetric response (CR) was determined by the percent changein blue color represented by the equationCR=[(PB_(initial)−P_(Bsample)/PB_(initial)]×100 where PB=blue color insample as determined using Adobe Photoshop, version 5, imaging software.Table of Abbreviations Abbreviation or Trade Name Description11-Bromo-1-undecanol Br(CH₂)₁₁OH 11-Bromoundecanoic acid Br(CH₂)₁₀C(O)OHTBDMSCl Tert-butyldimethylsilyl chloride Diyne-1Bis(trimethylsilyl)butadiyne Commercially available from Gelest;Tullytown, PA THF Tetrahydrofuran 1-Bromododecane Br(CH₂)₁₁CH₃ TBAFTetrabutylammonium fluoride HMPA Hexamethylphosphoramide1-Bromohexadecane Br(CH₂)₁₅CH₃ Methanesulfonyl chloride CH₂Cl₂Dichloromethane DMF Dimethylformamide oxalyl chloride ClCOCOCl DMPCDimyristoylphosphatidylcholine; commercially available from Avanti PolarLipids, Alabaster, Al. ATCC American Type Culture Collection PDCPyridinium dichromate DMAP 4-(dimethylamino)pyridine KAPA Potassium3-aminopropylamide prepared according to Abrams, S. R.; Shaw, A. C.Organic Syntheses, 1988, 66, 127-131. PP Polypropylene PE PolyethylenePTFE Polytetrafluoroethylene PVDF Polyvinylidenefluoride

Example 1

[0117] Preparation of HO(O)C(CH₂)₃C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃

[0118] Step 1: Preparation of HO(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃

[0119] In a glass reaction vessel, 600 milligrams of5,7-dodecadiyn-1,12-diol (HO(CH₂)₄C≡C—C≡C(CH₂)₄OH), 0.275 milliliters ofpyridine and 10 milliliters of THF were mixed. To this solution wasadded 676 milligrams of lauroyl chloride and the resulting mixture wasstirred for 15 hours. The mixture was then diluted with diethyl etherand washed with 0.1 N HCl and brine. The organic layer was separated,dried over MgSO₄, filtered and the solvent was removed to yield a whitesolid. The solid was purified over silica gel (gradient from 25% to 50%by volume ethyl acetate in hexanes) to yield 570 milligrams ofHO(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃ as a white solid.

[0120] Step 2: Preparation of HO(O)C(CH₂)₃C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃

[0121] In a glass reaction vessel, 377 milligrams ofHO(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃ prepared in Step 1 was dissolved in3 milliliters of DMF and 1.32 grams of PDC was added. The resultingmixture was stirred for 8 hours, and then worked up with water anddiethyl ether. The combined ether layers were dried over MgSO₄, filteredand the solvent was removed to yield a white solid. The solid waspurified over silica gel eluting with 25/74/1 of ethylacetate/hexanes/formic acid by volume to yield 0.21 grams ofHO(O)C(CH₂)₃C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₀CH₃ as a white solid.

Examples 2-5

[0122] Preparation of HO(O)C(CH₂)_(a-1)C≡C—C≡(CH₂)_(a)O(O)C(CH₂)_(b)CH₃

[0123] The same procedure described in Example 1 was followed using thediol and acid chloride in Step 1 shown in Table 2 to give the compoundswith the general structureHO(O)C(CH₂)_(a-1)C≡C—C≡C(CH₂)_(z)O(O)C(CH₂)_(b)CH₃ (a and b are definedin Table 2). TABLE 2 Exam- ple Diol, a value Acid Chloride, b value 2HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)₁₄C(O)Cl, b = a = 3 14 3HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₂C(O)Cl, b = a = 4 12 4HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₄C(O)Cl, b = a = 4 14 5HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₆C(O)Cl, b = a = 4 16

Example 6

[0124] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃

[0125] Step 1: Preparation of HO(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃

[0126] In a glass reaction vessel, 4.99 grams of5,7-dodecadiyn-1,12-diol (HO(CH₂)₄C≡C—C≡C(CH₂)₄OH), 2.2. grams ofpyridine and 50 milliliters of THF were mixed. To this solution wasadded 6.34 grams of myristol chloride and the resulting mixture wasstirred for 15 hours. The mixture was then diluted with diethyl etherand washed with 0.1 N HCl and brine. The organic layer was separated,dried over MgSO₄, filtered and the solvent was removed to yield a whitesolid. The solid was purified over silica gel (15% by volume of ethylacetate in dichloromethane to 100% theyl acetate gradient) to yield 5.0grams of HO(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃ as a white solid.

[0127] Step 2: Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃

[0128] In a sealable tube, 1.41 grams ofHO(CH₂)₃C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃ prepared in Step 1, 0.435 grams ofsuccinic anhydride, 13 milliliters of toluene and 0.106 grams of DMAPwere combined and the tube was sealed. The mixture was heated to 105° C.for 14.5 hours, the reaction was cooled to room temperature, 0.15milliliters of water was added, the tube was resealed and again heatedto 105° C. for 30 minutes. The mixture was then diluted with diethylether and washed with 0.1 N HCl and brine. The organic layer wasseparated, dried over MgSO₄, filtered and the solvent was removed toyield a white solid. The solid was purified over silica gel eluting with10/89/1 of ethyl acetate/dichloromethane/formic acid by volume to yield1.70 grams of HO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃ as awhite solid.

Examples 7-17

[0129] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)_(a)C≡C—C≡C(CH₂)_(a)O(O)C(CH₂)_(b)CH₃

[0130] The same procedure described in Example 6 was followed using thediol and acid chloride in Step 1 shown in Table 3 to give the compoundswith the general structureHO(O)C(CH₂)₂C(O)O(CH₂)_(a)C≡C—C≡C(CH₂)_(a)O(O)C(CH₂)_(b)CH₃ (a and b aredefined in Table 3). TABLE 3 Example Diol, a value Acid Chloride, bvalue 7 HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)₁₀C(O)Cl, a = 2 b = 10 8HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)₁₂C(O)Cl, a = 2 b = 12 9HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)₁₄C(O)Cl, a = 2 b = 14 10HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)₁₆C(O)Cl, a = 2 b = 16 11HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)₁₀C(O)Cl, a = 3 b = 10 12HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)₁₂C(O)Cl, a = 3 b = 12 13HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)₁₄C(O)Cl, a = 3 b = 14 14HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)₁₆C(O)Cl, a = 3 b = 16 15HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₀C(O)Cl, a = 4 b = 10 16HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₄C(O)Cl, a = 4 b = 14 17HO(CH₂)₄C≡C—C≡C(CH₂)₄OH, CH₃(CH₂)₁₆C(O)Cl, a = 4 b = 16

Example 18

[0131] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₅C≡C—C≡C(CH₂)₅O(O)C(CH₂)₁₀CH₃

[0132] Step 1: Preparation of HO(CH₂)₅C≡C—C≡C(CH₂)₅OH

[0133] HO(CH₂)₅C≡CH was prepared by the KAPA-promoted isomerization ofHOCH₂C═C(CH₂)₃CH₃ prepared according to Miller, J. G.; Oehlschlager, A.C. J.Org. Chem. 1984, 49, 2332-2338 or HO(CH₂)₂C═C(CH₂)₂CH₃(commercially available from GFS Chemicals; Powell, Ohio). Oxidativecoupling of HO(CH₂)₅C≡CH was carried out in a glass reaction vessel bydissolving 6.95 grams HO(CH₂)₅C≡CH in pyridine/methanol (2.0 mL/6.2 mL)and adding 307 grams of CuCl followed by stirring in the presence ofoxygen until all the starting material was consumed. The reactionmixture was worked up with diethyl ether and 4N HCl, the combinedorganic layers were dried over MgSO₄, filtered and concentrated.Recrystallization of the residue from 1/1 hexanes/tert-butyl methylether yielded 5.35 grams of HO(CH₂)₅C≡C—C≡C(CH₂)₅OH.

[0134] Step 2: Preparation of HO(CH₂)₅C≡C—C≡C(CH₂)₅O(O)C(CH₂)₁₀CH₃

[0135] The same procedure described in Example 6 Step 1 was followedexcept that instead of 5,7-dodecadiyn-1,12-diol the diol prepared inStep 1 above was used.

[0136] Step 3: Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₅C≡C—C≡C(CH₂)₅O(O)C(CH₂)₁₀CH₃

[0137] The same procedure described in Example 6 Step 2 was followed.

Examples 19-21

[0138] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₅C≡C—C≡C(CH₂)₅O(O)C(CH₂)_(b)CH₃

[0139] The same procedure described in Example 18 was followed using thediol and acid chloride in Step 2 shown in Table 4 to give the compoundswith the general structureHO(O)C(CH₂)₂C(O)O(CH₂)₅C≡C—C≡C(CH₂)₅O(O)C(CH₂)_(b)CH₃ (b is defined inTable 4). TABLE 4 Example Diol Acid Chloride, b value 19HO(CH₂)₅C≡C—C≡C(CH₂)₅OH CH₃(CH₂)₁₂C(O)Cl, b = 12 20HO(CH₂)₅C≡C—C≡C(CH₂)₅OH CH₃(CH₂)₁₄C(O)Cl, b = 14 21HO(CH₂)₅C≡C—C≡C(CH₂)₅OH CH₃(CH₂)₁₆C(O)Cl, b = 16

Examples 22-25

[0140] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₆C≡C—C≡C(CH₂)₆O(O)C(CH₂)_(b)CH₃

[0141] The same procedure described in Example 18 Step 1 was followed toprepare the diol HO(CH₂)₆C≡C—C≡C(CH₂)₆OH starting from 1-heptyne. Theremaining procedure for Example 18 was followed using the diol and acidchloride in Step 2 shown in Table 5 to give the compounds with thegeneral structure HO(O)C(CH₂)₂C(O)O(CH₂)₆C≡C—C≡C(CH₂)₆O(O)C(CH₂)_(b)CH₃(b is defined in Table 5). TABLE 5 Example Diol Acid Chloride, b value22 HO(CH₂)₆C≡C—C≡C(CH₂)₆OH CH₃(CH₂)₁₀C(O)Cl, b = 10 23HO(CH₂)₆C≡C—C≡C(CH₂)₆OH CH₃(CH₂)₁₂C(O)Cl, b = 12 24HO(CH₂)₆C≡C—C≡C(CH₂)₆OH CH₃(CH₂)₁₄C(O)Cl, b = 14 25HO(CH₂)₆C≡C—C≡C(CH₂)₆OH CH₃(CH₂)₁₆C(O)Cl, b = 16

Examples 26-29

[0142] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₇C≡C—C≡C(CH₂)₇O(O)C(CH₂)_(b)CH₃

[0143] The same procedure described in Example 18 Step 1 was followed toprepare the diol HO(CH₂)₇C≡C—C≡C(CH₂)₇OH staring from 1-octyne. Theremaining procedure for Example 18 was followed using the diol and acidchloride in Step 2 shown in Table 6 to give the compounds with thegeneral structure HO(O)C(CH₂)₂C(O)O(CH₂)₇C≡C—C≡C(CH₂)₇O(O)C(CH₂)_(b)CH₃(b is defined in Table 6). TABLE 6 Example Diol Acid Chloride 26HO(CH₂)₇C≡C—C≡C(CH₂)₇OH CH₃(CH₂)₁₀C(O)Cl, b = 10 27HO(CH₂)₇C≡C—C≡C(CH₂)₇OH CH₃(CH₂)₁₂C(O)Cl, b = 12 28HO(CH₂)₇C≡C—C≡C(CH₂)₇OH CH₃(CH₂)₁₆C(O)Cl, b = 16

Examples 29-32

[0144] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₉C≡C—C≡C(CH₂)₉O(O)C(CH₂)_(b)CH₃

[0145] The same procedure described in Example 18 Step 1 was followed toprepare the diol HO(CH₂)₉C═C—C═C(CH₂)₉OH staring from 1-decyne. Theremaining procedure for Example 18 was followed using the diol and acidchloride in Step 2 shown in Table 7 to give the compounds with thegeneral structure HO(O)C(CH₂)₂C(O)O(CH₂)₉C≡C—C≡C(CH₂)₉O(O)C(CH₂)_(b)CH₃(b is defined in Table 7). TABLE 7 Example Diol Acid Chloride, b value29 HO(CH₂)₉C≡C—C≡C(CH₂)₉OH CH₃(CH₂)₁₀C(O)Cl, b = 10 30HO(CH₂)₉C≡C—C≡C(CH₂)₉OH CH₃(CH₂)₁₂C(O)Cl, b = 12 31HO(CH₂)₉C≡C—C≡C(CH₂)₉OH CH₃(CH₂)₁₄C(O)Cl, b = 14 32HO(CH₂)₉C≡C—C≡C(CH₂)₉OH CH₃(CH₂)₁₆C(O)Cl, b = 16

Example 33

[0146] Preparation ofHO(O)C(CH₂)₃C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃

[0147] The same procedure described in Example 6 was followed exceptthat in Step 2 glutaric anhydride was used in place of succinicanhydride.

Example 34

[0148] Preparation of HO(O)CHC═CHC(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₄CH₃

[0149] The same procedure described in Example 6 was followed exceptthat in Step 1 CH₃(CH₂)₁₄C(O)Cl was used instead of CH₃(CH₂)₁₂C(O)Cl andin Step 2 maleic anhydride was used in place of succinic anhydride.

Example 35

[0150] Preparation ofHO(O)C(1,2-C₆H₄)C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(O)C(CH₂)₁₂CH₃

[0151] The same procedure described in Example 6 was followed exceptthat in Step 2 phthalic anhydride was used in place of succinicanhydride.

Example 36

[0152] Preparation of HO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(CH₂)₁₁CH₃

[0153] Step 1: Preparation of HO(CH₂)₄C≡C—C≡C(CH₂)₄O(CH₂)₁₁CH₃

[0154] In a glass reaction vessel, a suspension of 120 milligrams ofsodium hydride was prepared in 10 mL dry DMF and 972 milligrams of5,7-dodecadiyn-1,12-diol (HO(CH₂)₄C≡C—C≡C(CH₂)₄OH), was added. Afterstirring for 5 minutes, 1.31 grams of CH₃(CH₂)₁₁Br was added and theresulting mixture was stirred for 15 hours. The mixture was thenquenched by addition of saturated NH₄Cl solution and diluted with 100milliliters of diethyl ether. The organic layer was separated and washed3 times with brine, dried over MgSO₄, filtered and the solvent wasremoved to yield a yellow oil. The oil was purified over silica gel(25%-35% by volume of ethyl acetate in hexane gradient) to yield 606milligrams of HO(CH₂)₄C≡C—C≡C(CH₁₂)₄O(CH₂)₁₁CH₃ as a white solid.

[0155] Step 2: Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₁₂)₄O(CH₂)₁₁CH₃

[0156] In a sealable tube, 181 milligrams ofHO(CH₂)₄C═C—C═C(CH₂)₄O(CH₂)₁₁CH₃ prepared in Step 1, 63 milligrams ofsuccinic anhydride, 2 milliliters of toluene and 15 milligrams of DMAPwere combined and the tube was sealed. The mixture was heated to 110° C.for 16 hours, the reaction was cooled to room temperature, 3 drops ofwater were added, the tube was resealed and again heated to 1101° C. for30 minutes. The mixture was then diluted with diethyl ether and washedwith 0.1 N HCl and brine. The organic layer was separated, dried overMgSO₄, filtered and the solvent was removed to yield a white solid. Thesolid was purified over silica gel eluting with 10/89/1 of ethylacetate/dichloromethane/formic acid by volume to yieldHO(O)C(CH₂)₂C(O)O(CH₂)₄C≡C—C≡C(CH₂)₄O(CH₂)₁₁CH₃ as a white solid.

Examples 37-41

[0157] Preparation ofHO(O)C(CH₂)₂C(O)O(CH₂)_(a)C≡C—C≡C(CH₂)_(a)O(CH₂)_(b)CH₃

[0158] The same procedure described in Example 37 was followed using thediol and alkyl bromide in Step 1 shown in Table 8 to give the compoundswith the general structureHO(O)C(CH₂)₂C(O)O(CH₂)_(a)C≡C—C≡C(CH₂)_(a)O(CH₂)_(b)CH₃ (a and b aredefined in Table 8). TABLE 8 Exam- ple Diol, a value Alkyl Bromide, bvalue 37 HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)_(b)Br, a = 4 b = 13 38HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)_(b)Br, a = 4 b = 15 39HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)_(b)Br, a = 4 b = 17 40HO(CH₂)₂C≡C—C≡C(CH₂)₂OH, CH₃(CH₂)_(b)Br, a = 5 b = 11 41HO(CH₂)₃C≡C—C≡C(CH₂)₃OH, CH₃(CH₂)_(b)Br, a = 5 b = 13

Example 42

[0159] A sample of 10.1 milligrams of the compound prepared in Example 6was placed in a glass vessel and suspended in 5 milliliters ofisopropanol. The mixture was heated to boiling and 10 milliliters of 70°C. water was added. The resulting solution was boiled until thetemperature reached 95° C. indicating the nearly all of the isopropanolhad boiled off. The solution was cooled to room temperature and then to4° C. for 16 hours. A 2 milliliter aliquot of the solution was exposed254 nanometer light for 10 minutes, producing a dark blue colorindicative that polymerization had occurred.

Example 43

[0160] Detection of a Membrane Peptide in Solution

[0161] A sample of 1.0 milliMolar concentration of diacetylene monomerprepared as in Example 6 and DMPC (6:4) in Tris buffer (2 mM, pH=8.5)was prepared in a glass vessel, bath sonicated in a Bronson Model #1510bath sonicator (commercially available from VWR Scientific Products;West Chester, Pa.) for 30 minutes, and placed in a 4° C. refrigeratorfor about 16 hours. The sample was then filtered through a 1.2 μmsyringe filter, and polymerized by irradiating the sample beneath a 254nm UV lamp (commercially available from VWR Scientific Products; WestChester, Pa.) at a distance of 3 cm for 10 minutes. Polymerizationresults in the observation of a distinct, bluish-purple color.

[0162] The detection property of the solution was determined by theaddition of −80 μL of a polymyxin B sulfate solution (10,050 units/mL)to the sample. This resulted in a rapid colorimetric response that couldeasily be determined visually as a change from blue to red andquantified by UV-vis spectroscopy as a shift from peak absorbance at 640nm to a peak absorbance at 540 nm in spectra taken after one hour atroom temperature.

Example 44

[0163] Detection of a Membrane Peptide on a Substrate

[0164] The polymerized PDAIDMPC mixture prepared in Example 43 wascoated onto a piece of a reverse-phase C-18 silica gel plate. Thesolution was spotted on the plate and allowed to dry at room temperatureresulting in blue spots on the test plate. To test this solid state formof the sensor, a test solution (40 μL) of a polymyxin B sulfate (10,050units/mL) was added to the sample spot. This resulted in an immediate(<15 seconds) color change from blue to pink. A control test using onlymilli-Q water (50 μL) showed no color change.

Example 45

[0165] Detection of Peptide-Membrane Interactions in Solution

[0166] A mixture of diacetylene monomer from Example 6 and DMPC (6:4)was weighed into a vial and suspended in Tris buffer (2 mM, pH=8.5) toproduce a 1 mM concentration solution and a polymerized detectionsolution was prepared as in Example 43.

[0167] The detection property of the solution was determined by theaddition of −50 μL of a 1 mM mellitin solution to the sample. Thisresulted in a rapid calorimetric response that could easily bedetermined visually as a change from blue to red and quantified byUV-vis spectroscopy as a shift from peak absorbance at 640 nm to a peakabsorbance at 540 nm in spectra taken after one hour at roomtemperature.

Example 46

[0168] Detection of Peptide-Membrane Interactions on a Substrate

[0169] A test plate was made using the polymerized detector solution ofExample 45 using the same procedure as in Example 44. Several spots (40μl each) of the polymerized solutions were placed onto a piece of areverse-phase C-18 silica gel plate. The solutions were allowed to dryat room temperature resulting in blue spots. To test the substrates'ability to detect peptides, two samples containing 15 and 30 nanomolesof mellitin respectively were added to the different spots on the testplate. This resulted in an immediate (<15 seconds) color change fromblue to red. Control samples of milli-Q water spotted on separate testspots showed no color change.

Example 47

[0170] Detection of Interfacial Enzymatic Catalysis by Phospholipase A2in Solution

[0171] A detection solution was prepared as in Example 45. The detectionproperties of the solutions were determined by the addition of −50 μL ofa 75 μM Phospholipase A2 solution to each of the samples. This resultedin a colorimetric response that could easily be determined visually as achange from blue to red and quantified by UV-vis spectroscopy as a shiftfrom peak absorbance at 640 nm to a peak absorbance at 540 nm in spectrataken after one hour at room temperature.

Example 48

[0172] Attempted Detection of Peptide-Membrane Interactions usingCommercially Available Diacetylenic Monomers on a Substrate

[0173] Mixtures of commercially available 10,12-tricosadiynoic acid(available from GFS Chemicals; Powell, Ohio) and DMPC (6:4) were used tocreate a detector solution and test plate as in Example 44. Thepolymerization resulted in the observation of a distinct, bluish-purplecolor. On spotting the detector solution on the test plates some of thesolutions dried resulting in a color change to a red spot while othersamples dried to a blue colored spot.

[0174] To test the peptide-membrane interaction detection properties ofthe polymerized assemblies on a substrate, 30 nanomoles of mellitin(commercially available from Sigma Aldrich; St. Louis, Mo.) was added tothe spots that dried to a blue color. This resulted in either no colorchange or a splotchy appearance that reflected a CR less than 5%depending on the preparation of the solution.

Example 49

[0175] Attempted Detection of Peptide-Membrane Interactions usingCommercially Available Diacetylenic Monomers on a 2-D Substrate

[0176] Example 48 was repeated, except that to test the peptide-membraneinteraction detection properties of the polymerized assemblies on thesubstrate, 50 μL of a polymyxin B sulfate solution (10,050 units/mL) wasadded to each of the spots on the test plate. This resulted in asplotchy appearance that reflected a CR less than 5% of color changedepending on the preparation of the solution.

Example 50

[0177] Indirect Detection of E. coli on a Substrate

[0178] Test plates were made as in Example 46.

[0179] To test the detection of E. coli, a polymyxin B sulfate solution(10,050 units/mL) was added to a vial containing only milli-Q water andto a vial containing a suspension of E. coli [ATCC25922, ˜10⁹bacteria/mL in milli-Q water]. After allowing the two samples to sit for30 minutes, they were filtered through a 0.45 μm syringe filter and 40μL of each of the eluents was placed onto dried spots of the sensormixture. After 15 minutes the liquid was removed and the plates wereexamined. The sample with no E. coli and only polymyxin B showed a colorchange to red while those samples with both E. coli and polymyxin B showno dramatic color change.

Example 51

[0180] Detection of E. coli in a Biological Fluid on a Substrate

[0181] A mixture of diacetylene monomer as prepared in Example 6 andDMPC (6:4) was weighed into a vial and suspended in HEPES buffer (5 mM,pH=7.2) to produce a 1 mM solution. The solution was then probesonicated using a Model XL2020 probe sonicator (commercially availablefrom Misonix, Inc.; Farmington, N.Y.) for 1 minute at a power setting of5, and placed into a 4° C. refrigerator overnight (˜16 hours). Thesample was filtered through a 1.2 μm syringe filter and polymerizationof a stirring solution was achieved by irradiating the sample beneath a254 nm UV lamp at a distance of 3 cm for 20 minutes, resulting in theobservation of a blue color. Using a syringe, several spots (40 μl each)of the polymerized solution were placed onto a piece of a reverse-phaseC-18 silica gel plate. The spots were allowed to dry at room temperatureresulting in blue spots. To test the detection of E. coli, a polymyxin Bsulfate solution (10,050 units/mL) was added to a solution of humanurine and a solution of human urine contaminated with E. coli[ATCC25922, 109 bacteria/mL in milli-Q water]. After allowing thesamples to incubate for 30 minutes at 37° C., the samples were cooled toroom temperature and 40 mL of each eluent was placed onto a dried spotof the PDA/DMPC solution. After 40 minutes the liquid was removed andthe plates were examined. The spots showed a substantial color changefor the sample with no E. coli. and only polymyxin B present while thesample with E. coli and polymyxin B present exhibited little change incolor.

Example 52

[0182] Detection of Lipopolysaccharide on a Substrate

[0183] Mixtures of diacetylene monomer as prepared in Example 6 and DMPC(6:4) were weighed into vials and suspended in HEPES buffer (5 mM,pH=7.2) to produce a 1 mM solution. Test plates on a reverse-phase C-18silica gel plate were then prepared as in Example 43. To test thedetection of lipopolysaccharide, 1000 μL of a polymyxin B sulfatesolution (628 units/mL) was added to a 1 mL solution of endotoxin freewater and a 1 mL solution of endotoxin free water contaminated withlipopolysaccharide (10,000 units/mL). After allowing the samples toincubate for 30 minutes at 370° C., the samples were cooled to roomtemperature and 40 mL of each solution was placed onto a dried spot ofthe PDA/DMPC solution. After 60 minutes the liquid was removed and theplates were examined. The sample with no lipopolysaccharide showed acolor change to red while the solution with lipopolysaccharide andpolymyxin B showed no color change.

Example 53

[0184] Detection of Lipopolysaccharide on a 2-D Substrate Using Glycerol

[0185] Mixtures of diacetylene monomer as prepared in Example 6 andtetradecanoic acid 12-(4,4,-dihydroxy-butylroxy)-dodeca-5,7-diynyl ester(1:1) were weighed into vials and suspended in HEPES buffer (5 mM,pH=7.2) to produce a 1 mM solution. The solutions were then probesonicated using a Model XL2020 probe sonicator (commercially availablefrom Misonix, Inc.; Farmington, N.Y.) for 1 minute at a power setting of5, and then placed into a 4° C. refrigerator overnight (˜16 hours). Thesamples were filtered through a 1.2 μm syringe filter and polymerizationof a stirring solution was achieved by irradiating the sample beneath a254 nm UV lamp at a distance of 3 cm for 60 seconds, resulting in theobservation of an intense blue color. Using a syringe, several spots (40μl each) of the polymerized solutions were placed onto a piece of areverse-phase C-18 silica gel plate. The solutions were allowed to dryat room temperature resulting in blue spots. To test the detection oflipopolysaccharide, a polymyxin B sulfate solution (5025 units/mL) wasadded to a solution of endotoxin free water and a solution of endotoxinfree water contaminated with lipopolysaccharide. After allowing thesamples to incubate for 30 minutes at 37° C., the samples were cooled toroom temperature and 500 μL of each solution was placed onto a driedspot of the PDA/DMPC solution. After 15 minutes of gentle shaking, theliquid was removed and the plates were examined. This resulted in acalorimetric response that could easily be determined visually as achange from blue to red in the absence of lipopolysaccharide.

Example 54

[0186] Characterization of Substrates Amenable to Maintain the ActivePhase of the Polydiacetylene Assemblies

[0187] To determine the substrate characteristics identified in Table 1,surfaces were prepared in the following manner for evaluation. The goldsurfaces were prepared by evaporation of gold onto a chromium primedpolished silicon wafer. The resulting surface was highly reflective withan rms surface roughness less than 15 Ångstroms as measured by AtomicForce Microscopy (AFM) using Nanoscope Command Reference Manual Version4.42; Digital Instruments; Sections 12.5 and 12.6. The gold surfaceswere dusted prior to use using a dry nitrogen stream. The glass surfaceswere prepared by cleaning in an oxidizing bath (commercially availablefrom Nochromix) overnight, followed by copious rinsing in milli-Q wateruntil the rinse water could maintain a uniform sheet over the glasssurface without de-wetting. The glass surfaces were dusted prior to useusing a dry nitrogen stream.

[0188] Silicon wafer surfaces were used as received and dusted prior touse using a dry nitrogen stream. Surfaces modified with SAMs were formedby immersion of the surface in a solution nominally 1 mM in SAMconcentration. Either ethanol or chloroform were used as solvents.Immersion times were at least 24 hours, followed by extensive rinsingwith the neat solvent used during self-assembly. The samples were thenallowed to dry in a dry box overnight prior to the contact anglemeasurements.

[0189] All other surfaces were used as received. After preparation andtreatment, each sample was cut into several smaller pieces, some ofwhich were used for the contact angle measurements, and some for coatingwith the PDA solution. To coat the substrates, mixtures of diacetylenemonomer as prepared in Example 6 and DMPC (6:4) were weighed into vialsand suspended in HEPES buffer (5 mM, pH=7.2) to produce a 1 mM solution.The solutions were then probe sonicated using a Model XL2020 probesonicator (commercially available from Misonix, Inc.; Farmington, N.Y.)for 1 minute at a power setting of 5, and then placed into a 4° C.refrigerator overnight (˜16 hours). The samples were filtered through a1.2 μm syringe filter and a stirred solution was polymerized byirradiating the sample beneath a 254 nm UV lamp at a distance of 3 cmfor 20 minutes, resulting in the observation of a blue color. Using asyringe, several spots (40 μl each) of the polymerized solutions wereplaced onto a series of substrates specified in Table 1. The solutionswere allowed to dry at room temperature, and the resulting color of thespots was recorded.

[0190] The complete disclosures of the patents, patent documents, andpublications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousmodifications and alterations to this invention will become apparent tothose skilled in the art without departing from the scope and spirit ofthis invention. It should be understood that this invention is notintended to be unduly limited by the illustrative embodiments set forthherein and that such embodiments are presented by way of example only,with the scope of the invention intended to be limited only by theclaims.

What is claimed is:
 1. A colorimetric sensor for detecting an analyte,comprising: a receptor; and and a polymerized composition comprising atleast one compound of the formula

wherein R¹ comprises

R² comprises

R³, R⁸, R¹³, R²¹, R³¹, R²⁴, R³¹ and R³³ are independently C₁-C₂₀ alkyl;R⁴, R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵, and R³² are independentlyC₁-C₁₄ alkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently C₁-C₁₄alkylene, C₂-C₈ alkenylene, or C₆-C₁₃ arylene; R⁹ is C₁-C₁₄ alkylene or—NR³⁴—; R¹⁰, R¹², R²⁷, and R²⁹ are independently C₁-C₁₄ alkylene or(C₁-C₁₄ alkylene)-(C₂-C₈ arylene); R¹¹ and R²⁸ are independently C₂-C₃₀alkynyl; R¹⁷ is an ester-activating group; R²³ is C₆-C₁₃ arylene; R³⁰ isC₁-C₁₄ alkylene or —NR³⁶—; R³⁴, and R³⁶ are C₁-C₄ alkyl; p is 1-5; n is1-20; wherein R¹ and R² are not the same; wherein the receptor isincorporated into the polymerized composition to form a transducer; andwherein the transducer exhibits a color change when contacted with ananalyte.
 2. The sensor of claim 1, wherein R¹ is dodecyl or hexadecyl.3. The sensor of claim 1, wherein R³ is undecyl or pentadecyl.
 4. Thesensor of claim 1, wherein R²⁴ is methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, or dodecyl.
 5. The sensor of claim 1,wherein R³³ is dodecyl, tetradecyl, hexadecyl, or octadecyl.
 6. Thesensor of claim 1, wherein R⁴ is methylene, trimethylene, ortetramethylene.
 7. The sensor of claim 1, wherein R⁵ is ethylene ortrimethylene.
 8. The sensor of claim 1, wherein R⁷, R¹⁶, R¹⁹, R²⁰ andR²⁵ are independently ethylene, trimethylene, tetramethylene,pentamethylene, hexamethylene, heptamethylene, octamethylene, ornonamethylene.
 9. The sensor of claim 1, wherein R²⁰ is methylene,trimethylene, or tetramethylene.
 10. The sensor of claim 1, wherein R²²is ethylene, trimethylene, or tetramethylene.
 11. The sensor of claim 1,wherein R³² is ethylene, trimethylene, tetramethylene, pentamethylene,or hexamethylene.
 12. The sensor of claim 1, wherein R⁶, R¹⁵, R¹⁸, andR²⁶ are independently ethylene, trimethylene, ethenylene, or phenylene.13. The sensor of claim 1, wherein R⁸ and R³¹ are independently C₁-C₁₄alkyl.
 14. The sensor of claim 15, wherein R⁸ and R³¹ are independentlybutyl, pentyl or hexyl.
 15. The sensor of claim 1, wherein R⁹ and R³⁰are independently methylene, —NH—, —N(CH₂CH₃)—, or —N(CH₃)—.
 16. Thesensor of claim 1, wherein R¹⁰, R¹², R²⁷, and R²⁹ are independentlymethylene, ethylene, trimethylene, tetramethylene, —C(CH₃)₂—,—CH((CH₂)_(t-4)CH₃)—, or —CH₂-phenylene.
 17. The sensor of claim 1,wherein R¹¹ and R²⁸ are independently C₂-C₃₀ alkynyl having at least twocarbon-carbon triple bonds.
 18. The sensor of claim 19, wherein R¹¹ andR²⁸ are independently —(CH₂)₈—C≡C—C≡C—(CH₂)₉CH₃, or—(CH₂)₈—C≡C—C≡C—(CH₂)₁₁CH₃.
 19. The sensor of claim 1, wherein R¹³ isC₁-C₄ alkyl.
 20. The sensor of claim 1, wherein R¹⁴ is C₁-C₄ alkylene.21. The sensor of claim 1, wherein R¹⁷ is 2,5-dioxo-1-pyrrolidinyl. 22.The sensor of claim 1, wherein R²³ is phenylene.
 23. The sensor of claim1, wherein n is 1-20, 3-17, 6-14, or 9-11.
 24. The sensor of claim 1,wherein p is 1 or
 2. 25. The sensor of claim 1 wherein R¹ is

wherein R⁷ is ethylene, trimethylene, tetramethylene, pentamethylene,hexamethylene, heptamethylene, octamethylene, or nonamethylene, and R¹is ethylene, trimethylene, ethenylene, or phenylene; and wherein R² is

wherein R²⁰ is ethylene, trimethylene, tetramethylene, pentamethylene,hexamethylene, heptamethylene, octamethylene, or nonamethylene, andwherein R²¹ is undecyl, tridecyl, pentadecyl, heptadecyl; and wherein pis
 1. 26. The sensor of claim 25, wherein R¹ is

R⁷ is ethylene; and R² is

R²⁰ is tetramethylene, and wherein R²¹ is tridecyl; and p is
 1. 27. Thesensor of claim 1, wherein the receptor is selected from the groupconsisting of phospholipid and glycerol.
 28. The sensor of claim 1,wherein the transducer is dispersed in an aqueous solution.
 29. Thesensor of claim 1, wherein the transducer is coated on a substrate. 30.The sensor of claim 29, wherein the substrate exhibits a contact angleless than 50 degrees using methylene iodide.
 31. The sensor of claim 30,wherein the substrate is selected from the group consisting of silicagel plate, paper, glass, textured photo paper, gloss photo paper, andmicroporous film.
 32. The sensor of claim 1, wherein the receptor isintegrated into the polymerized composition by physical mixing.
 33. Thesensor of claim 1, wherein the receptor is covalently bonded to thepolymerized composition.
 34. A method for the detection of an analyte,comprising forming a colorimetric sensor, comprising a receptor and apolymerized composition of the formula

wherein R¹ comprises

R² comprises

R³, R⁸, R¹³, R²¹, R²⁴, R³¹ and R³³ are independently C₁-C₂₀ alkyl; R⁴,R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵, and R³² are independently C₁-C₁₄alkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently C₁-C₁₄ alkylene, C₂-C₈alkenylene, or C₆-C₁₃ arylene; R⁹ is C₁-C₁₄ alkylene or —NR³⁴—; R¹⁰,R¹², R²⁷, and R²⁹ are independently C₁-C₁₄ alkylene or (C₁-C₁₄alkylene)-(C₂-C₈ arylene); R¹¹ and R²⁸ are independently C₂-C₃₀ alkynyl;R¹⁷ is an ester-activating group; R²³ is C₆-C₁₃ arylene; R³⁰ is C₁-C₁₄alkylene or —NR³⁶—; R³⁴, and R³⁶ are C₃-C₄ alkyl; p is 1-5; n is 1-20; ,wherein R¹ and R² are not the same; wherein the receptor is incorporatedinto the polymerized composition to form a transducer capable ofexhibiting a color change; contacting the sensor with an analyte; andobserving a color change if the analyte is present.
 35. The sensor ofclaim 34, wherein the transducer is dispersed in an aqueous solution.36. The method of claim 34, wherein the transducer is coated on asubstrate.
 37. The method of claim 36, wherein the substrate exhibits acontact angle less than 50 degrees using methylene iodide.
 38. Thesensor of claim 30, wherein the substrate is selected from the groupconsisting of silica gel plate, paper, glass, textured photo paper,gloss photo paper, and microporous film.
 39. The sensor of claim 34,wherein the receptor is integrated into the polymerized composition byphysical mixing.
 40. The sensor of claim 34, wherein the receptor iscovalently bonded to the polymerized composition.
 41. A method for thedetection of an analyte, comprising forming a colorimetric sensor,comprising a receptor and a polymerized composition of the formula

wherein R¹ comprises

R² comprises

R³, R⁸, R¹³, R²¹, R²⁴, R³¹ and R³³ are independently C₁-C₂₀alkyl; R⁴,R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵, and R³² are independently C₁₋₁alkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently C₁-C₁₄ alkylene, C₂-C₈alkenylene, or C₆-C₁₃ arylene; R⁹ is C₁-C₁₄ alkylene or —NR³⁴—; R¹⁰,R¹², R²⁷, and R²⁹ are independently C₁-C₁₄ alkylene or (C₁-C₁₄alkylene)-(C₂-C₈ arylene); R¹¹ and R²⁸ are independently C₂-C₃₀ alkynyl;R¹⁷ is an ester-activating group; R²³ is C₆-C₁₃ arylene; R³⁰ is C₁-C₁₄alkylene or —NR³⁶—; R³⁴ and R¹⁶ are C₁-C₄ alkyl; p is 1-5; n is 1-20;wherein R¹ and R² are not the same; and wherein the receptor isincorporated into the polymerized composition to form a transducercapable of exhibiting a color change; contacting the transducer with ananalyte; contacting the transducer with a probe that has an affinity forboth the analyte and the receptor; and observing no color change if theanalyte is present.
 42. The method of claim 41, wherein the probe andanalyte are combined to form a mixture before contacting the transducer.43. The method of claim 41, wherein the transducer is dispersed in anaqueous solution.
 44. The method of claim 41, wherein the transducer iscoated on a substrate.
 45. The method of claim 44, wherein the substrateexhibits a contact angle less than 50 degrees using methylene iodide.46. The method of claim 45, wherein the substrate is selected from thegroup consisting of silica gel plate, paper, glass, textured photopaper, gloss photo paper, and microporous film.
 47. The method of claim41, wherein the receptor is integrated into the polymerized compositionby physical mixing.
 48. The method of claim 41, wherein the receptor iscovalently bonded to the polymerized composition.
 49. The method ofclaim 41, wherein the probe comprises a membrane disrupting peptide. 50.The method of claim 49, wherein the probe is selected from the groupconsisting of alamethicin, magainin, gramicidin, polymyxin B sulfate,and melittin.
 51. The method of claim 41, wherein the analyte isselected from the group consisting of gram-negative bacteria andendotoxin.
 52. A disposable kit for detecting the presence of ananalyte, comprising: one or more transducers comprising a receptor and apolymerized composition of the formula

wherein R¹ comprises

R² comprises

R³, R⁸, R¹³, R²¹, R²⁴, R³¹ and R³³ are independently C₁-C₂₀ alkyl; R⁴,R⁵, R⁷, R¹⁴, R¹⁶, R¹⁹, R²⁰, R²², R²⁵ and R³² are independentlyC₁-C₁₄alkylene; R⁶, R¹⁵, R¹⁸, and R²⁶ are independently C₁-C₁₄ alkylene,C₂-C₈ alkenylene, or C₆-C₁₃ arylene; R⁹ is C₁-C₁₄ alkylene or —NR³⁴—;R¹⁰, R¹², R²⁷, and R²⁹ are independently C₁-C₁₄ alkylene or (C₁-C₁₄alkylene)-(C₂-C₈ arylene); R¹¹ and R²⁸ are independently C₂-C₃₀ alkynyl;R¹⁷ is an ester-activating group; R²³ is C₆-C₁₃ arylene; R³⁰ is C₁-C₁₄alkylene or —NR³⁶—; R³⁴, and R³⁶ are C₁-C₄ alkyl; p is 1-5; n is 1-20;wherein R¹ and R² are not the same; and wherein the receptor isintegrated with the polymerized composition to form a transducer capableof exhibiting a color change; and a means for contacting the transducerwith an analyte.
 53. The kit of claim 52, further comprising one or moreprobes with an affinity to the receptor and one or more analytes;wherein the probe is physically separated from the transducer until thekit is used.
 54. The kit of claim 52, wherein one or more transducersare dispersed in an aqueous solution.
 55. The kit of claim 52, whereinone or more transducers are coated on a substrate to form an array. 56.The method of claim 55, wherein the substrate exhibits a contact angleless than 50 degrees using methylene iodide.
 57. The sensor of claim 56,wherein the substrate is selected from the group consisting of silicagel plate, paper, glass, textured photo paper, gloss photo paper, andmicroporous film.
 58. The kit of claim 52, wherein the receptor isintegrated into the polymerized composition by physical mixing.
 59. Thekit of claim 52, wherein the receptor is covalently bonded to thepolymerized composition.